Histoplasmosis is a common infection in the central United States as well as around the world. While it is usually a mile illness, it may be incapacitating or fatal in some patients, notably the immunocompromised. As the incidence of AIDS and the use of transplantation increase, this infection will assume increased importance, particularly in endemic areas. As the standard therapy for the infection is associated with significant toxicity, it would be clinically relevant to identify novel targets for chemotherapeutic intervention. Probing the genetic basis for fungal virulence is one avenue of investigation which is promising but which requires the ability to manipulate the genome of the fungus, including the capacity to move fungal genes between strains. Developing this ability is the basis for this proposal. My approach will be to isolate strains of Histoplasma capsulatum which exhibit resistance to benomyl and to oligomycin. The genes which encode this resistance, beta tubulin and ATP synthase subunit 9 respectively, will be isolated from the strains and used as dominant selectable markers in transformation experiments. Protoplast fusion will also be developed, using the benomyl- and oligomycin-resistant mutants as partners. Electrophoretic karyotyping of commonly used laboratory strains of H. capsulatum will be performed; the results will permit the mapping of transfected genes, the analysis of protoplast fusion products, and the study of the genomic organization of these strains. A unique, small (500 kilobase) chromosome DNA of other strains and characterized. Vector constructions employing portions of the small chromosome of G186B and repetitive DNA sequences will be tested by transformation. These studies will enhance our ability to study the basis for virulence of H. capsulatum. They will also shed light on the genomic organization of the fungus and the nature of inter-strain genomic variability. They will expand our understanding of large-DNA electrophoresis at the current upper limit of size for resolution, and thus provide a basis for studying genomic organization of other important lower eukaryotes.